Modification of Egg Plasma Membrane

这一提议的长期目标是确定将精子-卵子融合的初始信号转化为卵子对受精的各种反应的生化步骤。现已证实，鸡蛋的激活涉及蛋白酪氨酸激酶(PTKS)的作用，该蛋白参与启动钙瞬变，以及调节原核迁移和融合、启动DNA合成和有丝分裂。最近的研究表明，Fyn蛋白酪氨酸激酶在触发精子-卵子融合后的钙瞬变过程中发挥着重要作用。这项建议的目的是确定FYN活性在受精时被刺激的机制。以海胆卵为模型系统，我们认为Fyn激酶是由紧密偶联的磷酸酪氨酰磷酸酶激活的，该酶通过Tyr 527的去磷酸化来解除对Fyn的抑制。具体目的我将描述作用于海胆卵中Fyn的磷酸酶的特性。这种磷酸酶在卵子激活中的作用将通过显微注射实验来确定，该实验使用编码重组融合蛋白的mRNA和阻断抗体作为磷酸酶功能的特异性抑制剂。特殊目的II将描述FYN和该磷酸酶之间的相互作用部位。我们还将确定受精时激活的c-Fyn池的大小，并确定是否发生扩增事件。特殊目的III将检验c-Fyn在受精时被P-Tyr置换机制激活的假设。进一步的研究将试图设计一种基于P-Tyr置换机制的c-Fyn激动剂，本研究的结果将确定Fyn在受精时被激活的具体调控机制。这些信息将进一步了解卵子在受精时代谢途径和发育程序被激活的生化机制，从而增强我们促进人类成功发育的能力。

The long term objective of this proposal is to identify the biochemical steps which transduce the initial signal of sperm-egg fusion into the various responses of the egg to fertilization. It is now established that egg activation involves the action of protein tyrosine kinases (PTKS) which function to initiate the calcium transient, as well as regulate pronuclear migration and fusion, initiation of DNA synthesis, and mitosis. Recent studies have demonstrated that the Fyn protein tyrosine kinase plays a major role in triggering the calcium transient that follows sperm-egg fusion. The objective of this proposal is to identify the mechanism by which Fyn activity is stimulated at fertilization. Using the sea urchin egg as a model system, we propose that Fyn kinase is activated by a tightly coupled phosphotyrosyl phosphatase which relieves Fyn from inhibition via dephosphorylation of Tyr 527. Specific aim I will characterize the phosphatase that acts on Fyn in the sea urchin egg. The role of this phosphatase in egg activation will be determined through microinjection experiments using mRNA encoding recombinant fusion proteins and blocking antibodies as specific inhibitors of phosphatase function. Specific aim II will characterize the sites of interaction between Fyn and this phosphatase. We will also determine the size of the c-Fyn pool that is activated at fertilization and determine if an amplification event occurs. Specific Aim III will test the hypothesis that c-Fyn is activated by a P-Tyr displacement mechanism at fertilization. Further studies will attempt to design a c-Fyn agonist based on the P-Tyr displacement mechanism The results of this study will identify the specific regulatory mechanism by which Fyn is activated at fertilization. This information will further the understanding of the biochemical mechanism by which the metabolic pathways and developmental programs in the egg are activated at fertilization and thereby enhance our ability to promote successful development in humans.